Publications by authors named "Udo Reichl"

185 Publications

Cell-line screening and process development for a fusogenic oncolytic virus in small-scale suspension cultures.

Appl Microbiol Biotechnol 2022 Aug 29;106(13-16):4945-4961. Epub 2022 Jun 29.

Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, Sandtorstr. 1, 39106, Magdeburg, Germany.

Oncolytic viruses (OVs) represent a novel class of immunotherapeutics under development for the treatment of cancers. OVs that express a cognate or transgenic fusion protein is particularly promising as their enhanced intratumoral spread via syncytia formation can be a potent mechanism for tumor lysis and induction of antitumor immune responses. Rapid and efficient fusion of infected cells results in cell death before high titers are reached. Although this is an attractive safety feature, it also presents unique challenges for large-scale clinical-grade manufacture of OVs. Here we evaluate the use of four different suspension cell lines for the production of a novel fusogenic hybrid of vesicular stomatitis virus and Newcastle disease virus (rVSV-NDV). The candidate cell lines were screened for growth, metabolism, and virus productivity. Permissivity was evaluated based on extracellular infectious virus titers and cell-specific virus yields (CSVYs). For additional process optimizations, virus adaptation and multiplicity of infection (MOI) screenings were performed and confirmed in a 1 L bioreactor. BHK-21 and HEK293SF cells infected at concentrations of 2 × 10 cells/mL were identified as promising candidates for rVSV-NDV production, leading to infectious titers of 3.0 × 10 TCID/mL and 7.5 × 10 TCID/mL, and CSVYs of 153 and 9, respectively. Compared to the AGE1.CR.pIX reference produced in adherent cultures, oncolytic potency was not affected by production in suspension cultures and possibly even increased in cultures of HEK293SF and AGE1.CR.pIX. Our study describes promising suspension cell-based processes for efficient large-scale manufacturing of rVSV-NDV. KEY POINTS: • Cell contact-dependent oncolytic virus (OV) replicates in suspension cells. • Oncolytic potency is not encompassed during suspension cultivation. • Media composition, cell line, and MOI are critical process parameters for OV production. • The designed process is scalable and shows great promise for manufacturing clinical-grade material.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00253-022-12027-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9329169PMC
August 2022

Impact of Influenza A Virus Infection on Growth and Metabolism of Suspension MDCK Cells Using a Dynamic Model.

Metabolites 2022 Mar 12;12(3). Epub 2022 Mar 12.

Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse 1, 39106 Magdeburg, Germany.

Cell cultured-based influenza virus production is a viable option for vaccine manufacturing. In order to achieve a high concentration of viable cells, is requirement to have not only optimal process conditions, but also an active metabolism capable of intracellular synthesis of viral components. Experimental metabolic data collected in such processes are complex and difficult to interpret, for which mathematical models are an appropriate way to simulate and analyze the complex and dynamic interaction between the virus and its host cell. A dynamic model with 35 states was developed in this study to describe growth, metabolism, and influenza A virus production in shake flask cultivations of suspension Madin-Darby Canine Kidney (MDCK) cells. It considers cell growth (concentration of viable cells, mean cell diameters, volume of viable cells), concentrations of key metabolites both at the intracellular and extracellular level and virus titers. Using one set of parameters, the model accurately simulates the dynamics of mock-infected cells and correctly predicts the overall dynamics of virus-infected cells for up to 60 h post infection (hpi). The model clearly suggests that most changes observed after infection are related to cessation of cell growth and the subsequent transition to apoptosis and cell death. However, predictions do not cover late phases of infection, particularly for the extracellular concentrations of glutamate and ammonium after about 12 hpi. Results obtained from additional in silico studies performed indicated that amino acid degradation by extracellular enzymes resulting from cell lysis during late infection stages may contribute to this observed discrepancy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/metabo12030239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8950586PMC
March 2022

Absolute quantification of viral proteins during single-round replication of MDCK suspension cells.

J Proteomics 2022 05 1;259:104544. Epub 2022 Mar 1.

Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany; Bioprocess Engineering, Otto von Guericke University Magdeburg, Magdeburg, Germany.

Madin-Darby canine kidney (MDCK) cells are widely used in basic research and for the propagation of influenza A viruses (IAV) for vaccine production. To identify targets for antiviral therapies and to optimize vaccine manufacturing, a detailed understanding of the viral life cycle is important. This includes the characterization of virus entry, the synthesis of the various viral RNAs and proteins, the transfer of viral compounds in the cell and virus budding. In case quantitative information is available, the analysis can be complemented by mathematical modelling approaches. While comprehensive studies focusing on IAV entry as well as viral mRNA, vRNA and cRNA accumulation in the nucleus of cells have been performed, quantitative data regarding IAV protein synthesis and accumulation was mostly lacking. In this study, we present a mass spectrometry (MS)-based method to evaluate whether an absolute quantification of viral proteins is possible for single-round replication in suspension MDCK cells. Using influenza A/PR/8/34 (H1N1, RKI) as a model strain at a multiplicity of infection of ten, defined amounts of isotopically labelled peptides of synthetic origin of four IAV proteins (hemagglutinin, neuraminidase, nucleoprotein, matrix protein 1) were added as an internal standard before tryptic digestion of samples for absolute quantification (AQUA). The first intracellular protein detected was NP at 1 h post infection (hpi). A maximum extracellular concentration of 7.7E+12 copies/mL was achieved. This was followed by hemagglutinin (3 hpi, maximum 4.1E+12 copies/mL at 13 hpi), matrix protein 1 (5 hpi, maximum 2.2E+12 copies/mL at 13 hpi) and neuraminidase (5 hpi, 6.0E+11 copies/mL at 13 hpi). In sum, for the first time absolute IAV protein copy numbers were quantified by a MS-based method for infected MDCK cells providing important insights into viral protein dynamics during single-round virus replication. SIGNIFICANCE: Influenza A virus is a significant human pathogen worldwide. To improve therapies against influenza and overcome bottlenecks in vaccine production in cell culture, it is critical to gain a detailed understanding of the viral life cycle. In addition to qPCR-based models, this study will examine the dynamics of influenza virus proteins during infection of producer cells to gain initial insights into changes in absolute copy numbers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jprot.2022.104544DOI Listing
May 2022

High-Titer Hepatitis C Virus Production in a Scalable Single-Use High Cell Density Bioreactor.

Vaccines (Basel) 2022 Feb 7;10(2). Epub 2022 Feb 7.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital-Hvidovre, 2650 Hvidovre, Denmark.

Hepatitis C virus (HCV) infections pose a major public health burden due to high chronicity rates and associated morbidity and mortality. A vaccine protecting against chronic infection is not available but would be important for global control of HCV infections. In this study, cell culture-based HCV production was established in a packed-bed bioreactor (CelCradle™) aiming to further the development of an inactivated whole virus vaccine and to facilitate virological and immunological studies requiring large quantities of virus particles. HCV was produced in human hepatoma-derived Huh7.5 cells maintained in serum-free medium on days of virus harvesting. Highest virus yields were obtained when the culture was maintained with two medium exchanges per day. However, increasing the total number of cells in the culture vessel negatively impacted infectivity titers. Peak infectivity titers of up to 7.2 log focus forming units (FFU)/mL, accumulated virus yields of up to 5.9 × 10 FFU, and a cell specific virus yield of up to 41 FFU/cell were obtained from one CelCradle™. CelCradle™-derived and T flask-derived virus had similar characteristics regarding neutralization sensitivity and buoyant density. This packed-bed tide-motion system is available with larger vessels and may thus be a promising platform for large-scale HCV production.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/vaccines10020249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8880717PMC
February 2022

Critical Assessment of MetaProteome Investigation (CAMPI): a multi-laboratory comparison of established workflows.

Nat Commun 2021 12 15;12(1):7305. Epub 2021 Dec 15.

Département Médicaments et Technologies pour la Santé (DMTS), Université Paris Saclay, CEA, INRAE, SPI, 30200, Bagnols-sur-Cèze, France.

Metaproteomics has matured into a powerful tool to assess functional interactions in microbial communities. While many metaproteomic workflows are available, the impact of method choice on results remains unclear. Here, we carry out a community-driven, multi-laboratory comparison in metaproteomics: the critical assessment of metaproteome investigation study (CAMPI). Based on well-established workflows, we evaluate the effect of sample preparation, mass spectrometry, and bioinformatic analysis using two samples: a simplified, laboratory-assembled human intestinal model and a human fecal sample. We observe that variability at the peptide level is predominantly due to sample processing workflows, with a smaller contribution of bioinformatic pipelines. These peptide-level differences largely disappear at the protein group level. While differences are observed for predicted community composition, similar functional profiles are obtained across workflows. CAMPI demonstrates the robustness of present-day metaproteomics research, serves as a template for multi-laboratory studies in metaproteomics, and provides publicly available data sets for benchmarking future developments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-021-27542-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8674281PMC
December 2021

MPA_Pathway_Tool: User-Friendly, Automatic Assignment of Microbial Community Data on Metabolic Pathways.

Int J Mol Sci 2021 Oct 12;22(20). Epub 2021 Oct 12.

Bioprocess Engineering, Otto von Guericke University, Universitätsplatz 2, 39106 Magdeburg, Germany.

Taxonomic and functional characterization of microbial communities from diverse environments such as the human gut or biogas plants by multi-omics methods plays an ever more important role. Researchers assign all identified genes, transcripts, or proteins to biological pathways to better understand the function of single species and microbial communities. However, due to the versality of microbial metabolism and a still-increasing number of newly biological pathways, linkage to standard pathway maps such as the KEGG central carbon metabolism is often problematic. We successfully implemented and validated a new user-friendly, stand-alone web application, the MPA_Pathway_Tool. It consists of two parts, called 'Pathway-Creator' and 'Pathway-Calculator'. The 'Pathway-Creator' enables an easy set-up of user-defined pathways with specific taxonomic constraints. The 'Pathway-Calculator' automatically maps microbial community data from multiple measurements on selected pathways and visualizes the results. The MPA_Pathway_Tool is implemented in Java and ReactJS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms222010992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8539661PMC
October 2021

Continuous purification of influenza A virus particles using pseudo-affinity membrane chromatography.

J Biotechnol 2021 Dec 20;342:139-148. Epub 2021 Oct 20.

Bioprocess Engineering Group, Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstr. 1, 39106 Magdeburg, Germany; Chair of Bioprocess Engineering, Otto-von-Guericke University Magdeburg, Universität Platz 1, 39106 Magdeburg, Germany. Electronic address:

Robust and flexible continuous unit operations that enable the establishment of intensified bioprocesses is one of the most relevant trends in manufacturing of biopharmaceuticals, including virus-based products. Sulfated cellulose membrane adsorbers (SCMA) are one of the most promising matrices for chromatographic purification of virus particles, like influenza viruses. Here, a three 'column' periodical counter current set-up was used to continuously purify influenza A/PR/8/34 virus particles using SCMA in bind-elute mode. It was possible to recover 67.4% of the HA-activity and to remove 67.4% and 99.8% of the total protein and DNA, respectively. The performance of the continuous process operated over a total of 10 loops, was slightly inferior to was obtained in a comparable batch process. Nevertheless, it was possible to increase the effective usage of binding capacity to 80%, resulting on a productivity of 22.8 kHAU ml min. As a proof-of-principle, SCMA were successfully used as matrix for purification of cell-derived influenza virus particles, in continuous mode.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jbiotec.2021.10.003DOI Listing
December 2021

Synthetische Glykobiotechnologie.

Biospektrum (Heidelb) 2021 9;27(6):657-659. Epub 2021 Oct 9.

Abteilung Bioprozesstechnik (BPT), Max-Planck-Institut für Dynamik komplexer technischer Systeme, Sandtorstraße 1, D-39106 Magdeburg, Deutschland.

The field of synthetic glycobiotechnology encompasses the synthesis and modification of free carbohydrates and carbohydrates linked to biomolecules. Our group develops bio-catalytic processes for the synthesis of carbohydrate building blocks, so-called sugar nucleotides, and cell-free multi-enzyme cascades to tailor carbohydrates linked to proteins. The technology can eventually help to advance our understanding of the roles of specific carbohydrates in nutrition and medicine and contribute to human health and well-being.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12268-021-1659-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8501375PMC
October 2021

Cell-Free Multi-Enzyme Synthesis and Purification of Uridine Diphosphate Galactose.

Chembiochem 2022 01 14;23(2):e202100361. Epub 2021 Dec 14.

Max Planck Institute for Dynamics of Complex Technical Systems, Department of Bioprocess Engineering, Sandtorstrasse 1, 39106, Magdeburg, Germany.

High costs and low availability of UDP-galactose hampers the enzymatic synthesis of valuable oligosaccharides such as human milk oligosaccharides. Here, we report the development of a platform for the scalable, biocatalytic synthesis and purification of UDP-galactose. UDP-galactose was produced with a titer of 48 mM (27.2 g/L) in a small-scale batch process (200 μL) within 24 h using 0.02 g /g . Through in-situ ATP regeneration, the amount of ATP (0.6 mM) supplemented was around 240-fold lower than the stoichiometric equivalent required to achieve the final product yield. Chromatographic purification using porous graphic carbon adsorbent yielded UDP-galactose with a purity of 92 %. The synthesis was transferred to 1 L preparative scale production in a stirred tank bioreactor. To further reduce the synthesis costs here, the supernatant of cell lysates was used bypassing expensive purification of enzymes. Here, 23.4 g/L UDP-galactose were produced within 23 h with a synthesis yield of 71 % and a biocatalyst load of 0.05 g /g . The costs for substrates per gram of UDP-galactose synthesized were around 0.26 €/g.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cbic.202100361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9299652PMC
January 2022

Semi-continuous Propagation of Influenza A Virus and Its Defective Interfering Particles: Analyzing the Dynamic Competition To Select Candidates for Antiviral Therapy.

J Virol 2021 11 22;95(24):e0117421. Epub 2021 Sep 22.

Max Planck Institute for Dynamics of Complex Technical Systemsgrid.419517.f, Bioprocess Engineering, Magdeburg, Germany.

Defective interfering particles (DIPs) of influenza A virus (IAV) are naturally occurring mutants that have an internal deletion in one of their eight viral RNA (vRNA) segments, rendering them propagation-incompetent. Upon coinfection with infectious standard virus (STV), DIPs interfere with STV replication through competitive inhibition. Thus, DIPs are proposed as potent antivirals for treatment of the influenza disease. To select corresponding candidates, we studied generation of DIPs and propagation competition between different defective interfering (DI) vRNAs in an STV coinfection scenario in cell culture. A small-scale two-stage cultivation system that allows long-term semi-continuous propagation of IAV and its DIPs was used. Strong periodic oscillations in virus titers were observed due to the dynamic interaction of DIPs and STVs. Using next-generation sequencing, we detected a predominant formation and accumulation of DI vRNAs on the polymerase-encoding segments. Short DI vRNAs accumulated to higher fractions than longer ones, indicating a replication advantage, yet an optimum fragment length was observed. Some DI vRNAs showed breaking points in a specific part of their bundling signal (belonging to the packaging signal), suggesting its dispensability for DI vRNA propagation. Over a total cultivation time of 21 days, several individual DI vRNAs accumulated to high fractions, while others decreased. Using reverse genetics for IAV, purely clonal DIPs derived from highly replicating DI vRNAs were generated. We confirm that these DIPs exhibit a superior interfering efficacy compared to DIPs derived from lowly accumulated DI vRNAs and suggest promising candidates for efficacious antiviral treatment. Defective interfering particles (DIPs) emerge naturally during viral infection and typically show an internal deletion in the viral genome. Thus, DIPs are propagation-incompetent. Previous research suggests DIPs as potent antiviral compounds for many different virus families due to their ability to interfere with virus replication by competitive inhibition. For instance, the administration of influenza A virus (IAV) DIPs resulted in a rescue of mice from an otherwise lethal IAV dose. Moreover, no apparent toxic effects were observed when only DIPs were administered to mice and ferrets. IAV DIPs show antiviral activity against many different IAV strains, including pandemic and highly pathogenic avian strains, and even against nonhomologous viruses, such as SARS-CoV-2, by stimulation of innate immunity. Here, we used a cultivation/infection system, which exerted selection pressure toward accumulation of highly competitive IAV DIPs. These DIPs showed a superior interfering efficacy , and we suggest them for effective antiviral therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.01174-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8610589PMC
November 2021

Cell culture-based production of defective interfering influenza A virus particles in perfusion mode using an alternating tangential flow filtration system.

Appl Microbiol Biotechnol 2021 Oct 14;105(19):7251-7264. Epub 2021 Sep 14.

Chair of Bioprocess Engineering, Otto-Von-Guericke-University Magdeburg, Magdeburg, Germany.

Respiratory diseases including influenza A virus (IAV) infections represent a major threat to human health. While the development of a vaccine requires a lot of time, a fast countermeasure could be the use of defective interfering particles (DIPs) for antiviral therapy. IAV DIPs are usually characterized by a large internal deletion in one viral RNA segment. Consequentially, DIPs can only propagate in presence of infectious standard viruses (STVs), compensating the missing gene function. Here, they interfere with and suppress the STV replication and might act "universally" against many IAV subtypes. We recently reported a production system for purely clonal DIPs utilizing genetically modified cells. In the present study, we established an automated perfusion process for production of a DIP, called DI244, using an alternating tangential flow filtration (ATF) system for cell retention. Viable cell concentrations and DIP titers more than 10 times higher than for a previously reported batch cultivation were observed. Furthermore, we investigated a novel tubular cell retention device for its potential for continuous virus harvesting into the permeate. Very comparable performances to typically used hollow fiber membranes were found during the cell growth phase. During the virus replication phase, the tubular membrane, in contrast to the hollow fiber membrane, allowed 100% of the produced virus particles to pass through. To our knowledge, this is the first time a continuous virus harvest was shown for a membrane-based perfusion process. Overall, the process established offers interesting possibilities for advanced process integration strategies for next-generation virus particle and virus vector manufacturing.Key points• An automated perfusion process for production of IAV DIPs was established.• DIP titers of 7.40E + 9 plaque forming units per mL were reached.• A novel tubular cell retention device enabled continuous virus harvesting.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00253-021-11561-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8437742PMC
October 2021

A high cell density perfusion process for Modified Vaccinia virus Ankara production: Process integration with inline DNA digestion and cost analysis.

Biotechnol Bioeng 2021 12 23;118(12):4720-4734. Epub 2021 Sep 23.

Bioprocess Engineering Group, Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany.

By integrating continuous cell cultures with continuous purification methods, process yields and product quality attributes have been improved over the last 10 years for recombinant protein production. However, for the production of viral vectors such as Modified Vaccinia virus Ankara (MVA), no such studies have been reported although there is an increasing need to meet the requirements for a rising number of clinical trials against infectious or neoplastic diseases. Here, we present for the first time a scalable suspension cell (AGE1.CR.pIX cells) culture-based perfusion process in bioreactors integrating continuous virus harvesting through an acoustic settler with semi-continuous chromatographic purification. This allowed obtaining purified MVA particles with a space-time yield more than 600% higher for the integrated perfusion process (1.05 × 10 TCID /L /day) compared to the integrated batch process. Without further optimization, purification by membrane-based steric exclusion chromatography resulted in an overall product recovery of 50.5%. To decrease the level of host cell DNA before chromatography, a novel inline continuous DNA digestion step was integrated into the process train. A detailed cost analysis comparing integrated production in batch versus production in perfusion mode showed that the cost per dose for MVA was reduced by nearly one-third using this intensified small-scale process.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/bit.27937DOI Listing
December 2021

Multiscale model of defective interfering particle replication for influenza A virus infection in animal cell culture.

PLoS Comput Biol 2021 09 7;17(9):e1009357. Epub 2021 Sep 7.

Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany.

Cell culture-derived defective interfering particles (DIPs) are considered for antiviral therapy due to their ability to inhibit influenza A virus (IAV) production. DIPs contain a large internal deletion in one of their eight viral RNAs (vRNAs) rendering them replication-incompetent. However, they can propagate alongside their homologous standard virus (STV) during infection in a competition for cellular and viral resources. So far, experimental and modeling studies for IAV have focused on either the intracellular or the cell population level when investigating the interaction of STVs and DIPs. To examine these levels simultaneously, we conducted a series of experiments using highly different multiplicities of infections for STVs and DIPs to characterize virus replication in Madin-Darby Canine Kidney suspension cells. At several time points post infection, we quantified virus titers, viable cell concentration, virus-induced apoptosis using imaging flow cytometry, and intracellular levels of vRNA and viral mRNA using real-time reverse transcription qPCR. Based on the obtained data, we developed a mathematical multiscale model of STV and DIP co-infection that describes dynamics closely for all scenarios with a single set of parameters. We show that applying high DIP concentrations can shut down STV propagation completely and prevent virus-induced apoptosis. Interestingly, the three observed viral mRNAs (full-length segment 1 and 5, defective interfering segment 1) accumulated to vastly different levels suggesting the interplay between an internal regulation mechanism and a growth advantage for shorter viral RNAs. Furthermore, model simulations predict that the concentration of DIPs should be at least 10000 times higher than that of STVs to prevent the spread of IAV. Ultimately, the model presented here supports a comprehensive understanding of the interactions between STVs and DIPs during co-infection providing an ideal platform for the prediction and optimization of vaccine manufacturing as well as DIP production for therapeutic use.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pcbi.1009357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8448327PMC
September 2021

Cell-Free Glycoengineering of the Recombinant SARS-CoV-2 Spike Glycoprotein.

Front Bioeng Biotechnol 2021 16;9:699025. Epub 2021 Aug 16.

Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, Magdeburg, Germany.

The baculovirus-insect cell expression system is readily utilized to produce viral glycoproteins for research as well as for subunit vaccines and vaccine candidates, for instance against SARS-CoV-2 infections. However, the glycoforms of recombinant proteins derived from this expression system are inherently different from mammalian cell-derived glycoforms with mainly complex-type glycans attached, and the impact of these differences in protein glycosylation on the immunogenicity is severely under investigated. This applies also to the SARS-CoV-2 spike glycoprotein, which is the antigen target of all licensed vaccines and vaccine candidates including virus like particles and subunit vaccines that are variants of the spike protein. Here, we expressed the transmembrane-deleted human -1,2 N-acetlyglucosamintransferases I and II (MGAT1ΔTM and MGAT2ΔTM) and the -1,4-galactosyltransferase (GalTΔTM) in to remodel the -glycans of a recombinant SARS-CoV-2 spike glycoprotein derived from insect cells. In a cell-free sequential one-pot reaction, fucosylated and afucosylated paucimannose-type -glycans were converted to complex-type galactosylated -glycans. In the future, this glycoengineering approach can be used to efficiently generate a wide range of -glycans on antigens considered as vaccine candidates for animal trials and preclinical testing to better characterize the impact of -glycosylation on immunity and to improve the efficacy of protein subunit vaccines.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fbioe.2021.699025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8415157PMC
August 2021

Combining functional metagenomics and glycoanalytics to identify enzymes that facilitate structural characterization of sulfated N-glycans.

Microb Cell Fact 2021 Aug 21;20(1):162. Epub 2021 Aug 21.

New England Biolabs, Ipswich, MA, 01938, USA.

Background: Sulfate modification of N-glycans is important for several biological functions such as clearance of pituitary hormones or immunoregulation. Yet, the prevalence of this N-glycan modification and its functions remain largely unexplored. Characterization of N-glycans bearing sulfate modifications is hampered in part by a lack of enzymes that enable site-specific detection of N-glycan sulfation. In this study, we used functional metagenomic screening to identify enzymes that act upon sulfated N-acetylglucosamine (GlcNAc). Using multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) -based glycoanalysis we proved their ability to act upon GlcNAc-6-SO on N-glycans.

Results: Our screen identified a sugar-specific sulfatase that specifically removes sulfate from GlcNAc-6-SO when it is in a terminal position on an N-glycan. Additionally, in the absence of calcium, this sulfatase binds to the sulfated glycan but does not remove the sulfate group, suggesting it could be used for selective isolation of sulfated N-glycans. Further, we describe isolation of a sulfate-dependent hexosaminidase that removes intact GlcNAc-6-SO (but not asulfated GlcNAc) from a terminal position on N-glycans. Finally, the use of these enzymes to detect the presence of sulfated N-glycans by xCGE-LIF is demonstrated.

Conclusion: The present study demonstrates the feasibility of using functional metagenomic screening combined with glycoanalytics to discover enzymes that act upon chemical modifications of glycans. The discovered enzymes represent new specificities that can help resolve the presence of GlcNAc-6-SO in N-glycan structural analyses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12934-021-01652-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8379841PMC
August 2021

Antiviral Activity of Influenza A Virus Defective Interfering Particles against SARS-CoV-2 Replication In Vitro through Stimulation of Innate Immunity.

Cells 2021 07 11;10(7). Epub 2021 Jul 11.

Immune Regulation Group, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19) emerged in late 2019 and resulted in a devastating pandemic. Although the first approved vaccines were already administered by the end of 2020, worldwide vaccine availability is still limited. Moreover, immune escape variants of the virus are emerging against which the current vaccines may confer only limited protection. Further, existing antivirals and treatment options against COVID-19 show only limited efficacy. Influenza A virus (IAV) defective interfering particles (DIPs) were previously proposed not only for antiviral treatment of the influenza disease but also for pan-specific treatment of interferon (IFN)-sensitive respiratory virus infections. To investigate the applicability of IAV DIPs as an antiviral for the treatment of COVID-19, we conducted in vitro co-infection experiments with cell culture-derived DIPs and the IFN-sensitive SARS-CoV-2 in human lung cells. We show that treatment with IAV DIPs leads to complete abrogation of SARS-CoV-2 replication. Moreover, this inhibitory effect was dependent on janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling. Further, our results suggest boosting of IFN-induced antiviral activity by IAV DIPs as a major contributor in suppressing SARS-CoV-2 replication. Thus, we propose IAV DIPs as an effective antiviral agent for treatment of COVID-19, and potentially also for suppressing the replication of new variants of SARS-CoV-2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cells10071756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8303422PMC
July 2021

Towards integrated production of an influenza A vaccine candidate with MDCK suspension cells.

Biotechnol Bioeng 2021 10 20;118(10):3996-4013. Epub 2021 Jul 20.

Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany.

Seasonal influenza epidemics occur both in northern and southern hemispheres every year. Despite the differences in influenza virus surface antigens and virulence of seasonal subtypes, manufacturers are well-adapted to respond to this periodical vaccine demand. Due to decades of influenza virus research, the development of new influenza vaccines is relatively straight forward. In similarity with the ongoing coronavirus disease 2019 pandemic, vaccine manufacturing is a major bottleneck for a rapid supply of the billions of doses required worldwide. In particular, egg-based vaccine production would be difficult to schedule and shortages of other egg-based vaccines with high demands also have to be anticipated. Cell culture-based production systems enable the manufacturing of large amounts of vaccines within a short time frame and expand significantly our options to respond to pandemics and emerging viral diseases. In this study, we present an integrated process for the production of inactivated influenza A virus vaccines based on a Madin-Darby Canine Kidney (MDCK) suspension cell line cultivated in a chemically defined medium. Very high titers of 3.6 log (HAU/100 µl) were achieved using fast-growing MDCK cells at concentrations up to 9.5 × 10 cells/ml infected with influenza A/PR/8/34 H1N1 virus in 1 L stirred tank bioreactors. A combination of membrane-based steric-exclusion chromatography followed by pseudo-affinity chromatography with a sulfated cellulose membrane adsorber enabled full recovery for the virus capture step and up to 80% recovery for the virus polishing step. Purified virus particles showed a homogenous size distribution with a mean diameter of 80 nm. Based on a monovalent dose of 15 µg hemagglutinin (single-radial immunodiffusion assay), the level of total protein and host cell DNA was 58 µg and 10 ng, respectively. Furthermore, all process steps can be fully scaled up to industrial quantities for commercial manufacturing of either seasonal or pandemic influenza virus vaccines. Fast production of up to 300 vaccine doses per liter within 4-5 days makes this process competitive not only to other cell-based processes but to egg-based processes as well.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/bit.27876DOI Listing
October 2021

SARS-CoV-2 Production in a Scalable High Cell Density Bioreactor.

Vaccines (Basel) 2021 Jun 29;9(7). Epub 2021 Jun 29.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital-Hvidovre, 2650 Hvidovre, Denmark.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has demonstrated the value of pursuing different vaccine strategies. Vaccines based on whole viruses, a widely used vaccine technology, depend on efficient virus production. This study aimed to establish SARS-CoV-2 production in the scalable packed-bed CelCradle 500-AP bioreactor. CelCradle 500-AP bottles with 0.5 L working volume and 5.5 g BioNOC™ II carriers were seeded with 1.5 × 10 Vero (WHO) cells, approved for vaccine production, in animal component-free medium and infected at a multiplicity of infection of 0.006 at a total cell number of 2.2-2.5 × 10 cells/bottle seven days post cell seeding. Among several tested conditions, two harvests per day and a virus production temperature of 33 °C resulted in the highest virus yield with a peak SARS-CoV-2 infectivity titer of 7.3 log 50% tissue culture infectious dose (TCID)/mL at 72 h post-infection. Six harvests had titers of ≥6.5 log TCID/mL, and a total of 10.5 log TCID were produced in ~5 L. While trypsin was reported to enhance virus spread in cell culture, addition of 0.5% recombinant trypsin after infection did not improve virus yields. Overall, we demonstrated successful animal component-free production of SARS-CoV-2 in well-characterized Vero (WHO) cells in a scalable packed-bed bioreactor.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/vaccines9070706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8310283PMC
June 2021

Fecal Metaproteomics Reveals Reduced Gut Inflammation and Changed Microbial Metabolism Following Lifestyle-Induced Weight Loss.

Biomolecules 2021 05 12;11(5). Epub 2021 May 12.

Bioprocess Engineering, Otto von Guericke University, Universitätsplatz 2, 39106 Magdeburg, Germany.

Gut microbiota-mediated inflammation promotes obesity-associated low-grade inflammation, which represents a hallmark of metabolic syndrome. To investigate if lifestyle-induced weight loss (WL) may modulate the gut microbiome composition and its interaction with the host on a functional level, we analyzed the fecal metaproteome of 33 individuals with metabolic syndrome in a longitudinal study before and after lifestyle-induced WL in a well-defined cohort. The 6-month WL intervention resulted in reduced BMI (-13.7%), improved insulin sensitivity (HOMA-IR, -46.1%), and reduced levels of circulating hsCRP (-39.9%), indicating metabolic syndrome reversal. The metaprotein spectra revealed a decrease of human proteins associated with gut inflammation. Taxonomic analysis revealed only minor changes in the bacterial composition with an increase of the families Desulfovibrionaceae, Leptospiraceae, Syntrophomonadaceae, Thermotogaceae and Verrucomicrobiaceae. Yet we detected an increased abundance of microbial metaprotein spectra that suggest an enhanced hydrolysis of complex carbohydrates. Hence, lifestyle-induced WL was associated with reduced gut inflammation and functional changes of human and microbial enzymes for carbohydrate hydrolysis while the taxonomic composition of the gut microbiome remained almost stable. The metaproteomics workflow has proven to be a suitable method for monitoring inflammatory changes in the fecal metaproteome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/biom11050726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8150863PMC
May 2021

Cell culture-based production and in vivo characterization of purely clonal defective interfering influenza virus particles.

BMC Biol 2021 05 3;19(1):91. Epub 2021 May 3.

Otto-von-Guericke-University Magdeburg, Chair of Bioprocess Engineering, Magdeburg, Germany.

Background: Infections with influenza A virus (IAV) cause high morbidity and mortality in humans. Additional to vaccination, antiviral drugs are a treatment option. Besides FDA-approved drugs such as oseltamivir or zanamivir, virus-derived defective interfering (DI) particles (DIPs) are considered promising new agents. IAV DIPs typically contain a large internal deletion in one of their eight genomic viral RNA (vRNA) segments. Consequently, DIPs miss the genetic information necessary for replication and can usually only propagate by co-infection with infectious standard virus (STV), compensating for their defect. In such a co-infection scenario, DIPs interfere with and suppress STV replication, which constitutes their antiviral potential.

Results: In the present study, we generated a genetically engineered MDCK suspension cell line for production of a purely clonal DIP preparation that has a large deletion in its segment 1 (DI244) and is not contaminated with infectious STV as egg-derived material. First, the impact of the multiplicity of DIP (MODIP) per cell on DI244 yield was investigated in batch cultivations in shake flasks. Here, the highest interfering efficacy was observed for material produced at a MODIP of 1E-2 using an in vitro interference assay. Results of RT-PCR suggested that DI244 material produced was hardly contaminated with other defective particles. Next, the process was successfully transferred to a stirred tank bioreactor (500 mL working volume) with a yield of 6.0E+8 PFU/mL determined in genetically modified adherent MDCK cells. The produced material was purified and concentrated about 40-fold by membrane-based steric exclusion chromatography (SXC). The DI244 yield was 92.3% with a host cell DNA clearance of 97.1% (99.95% with nuclease digestion prior to SXC) and a total protein reduction of 97.2%. Finally, the DIP material was tested in animal experiments in D2(B6).A2G-Mx1 mice. Mice infected with a lethal dose of IAV and treated with DIP material showed a reduced body weight loss and all animals survived.

Conclusion: In summary, experiments not only demonstrated that purely clonal influenza virus DIP preparations can be obtained with high titers from animal cell cultures but confirmed the potential of cell culture-derived DIPs as an antiviral agent.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12915-021-01020-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8091782PMC
May 2021

Site-specific N-glycosylation analysis of animal cell culture-derived Zika virus proteins.

Sci Rep 2021 03 4;11(1):5147. Epub 2021 Mar 4.

Bioprocess Engineering Group, Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany.

Here, we present for the first time, a site-specific N-glycosylation analysis of proteins from a Brazilian Zika virus (ZIKV) strain. The virus was propagated with high yield in an embryo-derived stem cell line (EB66, Valneva SE), and concentrated by g-force step-gradient centrifugation. Subsequently, the sample was proteolytically digested with different enzymes, measured via a LC-MS/MS-based workflow, and analyzed in a semi-automated way using the in-house developed glyXtool software. The viral non-structural protein 1 (NS1) was glycosylated exclusively with high-mannose structures on both potential N-glycosylation sites. In case of the viral envelope (E) protein, no specific N-glycans could be identified with this method. Nevertheless, N-glycosylation could be proved by enzymatic de-N-glycosylation with PNGase F, resulting in a strong MS-signal of the former glycopeptide with deamidated asparagine at the potential N-glycosylation site N444. This confirmed that this site of the ZIKV E protein is highly N-glycosylated but with very high micro-heterogeneity. Our study clearly demonstrates the progress made towards site-specific N-glycosylation analysis of viral proteins, i.e. for Brazilian ZIKV. It allows to better characterize viral isolates, and to monitor glycosylation of major antigens. The method established can be applied for detailed studies regarding the impact of protein glycosylation on antigenicity and human pathogenicity of many viruses including influenza virus, HIV and corona virus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-021-84682-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933209PMC
March 2021

Comprehensive N-glycosylation analysis of the influenza A virus proteins HA and NA from adherent and suspension MDCK cells.

FEBS J 2021 08 15;288(16):4869-4891. Epub 2021 Mar 15.

Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany.

Glycosylation is considered as a critical quality attribute for the production of recombinant biopharmaceuticals such as hormones, blood clotting factors, or monoclonal antibodies. In contrast, glycan patterns of immunogenic viral proteins, which differ significantly between the various expression systems, are hardly analyzed yet. The influenza A virus (IAV) proteins hemagglutinin (HA) and neuraminidase (NA) have multiple N-glycosylation sites, and alteration of N-glycan micro- and macroheterogeneity can have strong effects on virulence and immunogenicity. Here, we present a versatile and powerful glycoanalytical workflow that enables a comprehensive N-glycosylation analysis of IAV glycoproteins. We challenged our workflow with IAV (A/PR/8/34 H1N1) propagated in two closely related Madin-Darby canine kidney (MDCK) cell lines, namely an adherent MDCK cell line and its corresponding suspension cell line. As expected, N-glycan patterns of HA and NA from virus particles produced in both MDCK cell lines were similar. Detailed analysis of the HA N-glycan microheterogeneity showed an increasing variability and a higher complexity for N-glycosylation sites located closer to the head region of the molecule. In contrast, NA was found to be exclusively N-glycosylated at site N73. Almost all N-glycan structures were fucosylated. Furthermore, HA and NA N-glycan structures were exclusively hybrid- and complex-type structures, to some extent terminated with alpha-linked galactose(s) but also with blood group H type 2 and blood group A epitopes. In contrast to the similarity of the overall glycan pattern, differences in the relative abundance of individual structures were identified. This concerned, in particular, oligomannose-type, alpha-linked galactose, and multiantennary complex-type N-glycans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/febs.15787DOI Listing
August 2021

Tracking changes in adaptation to suspension growth for MDCK cells: cell growth correlates with levels of metabolites, enzymes and proteins.

Appl Microbiol Biotechnol 2021 Mar 13;105(5):1861-1874. Epub 2021 Feb 13.

Bioprocess Engineering, Otto von Guericke University Magdeburg, Magdeburg, Germany.

Adaptations of animal cells to growth in suspension culture concern in particular viral vaccine production, where very specific aspects of virus-host cell interaction need to be taken into account to achieve high cell specific yields and overall process productivity. So far, the complexity of alterations on the metabolism, enzyme, and proteome level required for adaptation is only poorly understood. In this study, for the first time, we combined several complex analytical approaches with the aim to track cellular changes on different levels and to unravel interconnections and correlations. Therefore, a Madin-Darby canine kidney (MDCK) suspension cell line, adapted earlier to growth in suspension, was cultivated in a 1-L bioreactor. Cell concentrations and cell volumes, extracellular metabolite concentrations, and intracellular enzyme activities were determined. The experimental data set was used as the input for a segregated growth model that was already applied to describe the growth dynamics of the parental adherent cell line. In addition, the cellular proteome was analyzed by liquid chromatography coupled to tandem mass spectrometry using a label-free protein quantification method to unravel altered cellular processes for the suspension and the adherent cell line. Four regulatory mechanisms were identified as a response of the adaptation of adherent MDCK cells to growth in suspension. These regulatory mechanisms were linked to the proteins caveolin, cadherin-1, and pirin. Combining cell, metabolite, enzyme, and protein measurements with mathematical modeling generated a more holistic view on cellular processes involved in the adaptation of an adherent cell line to suspension growth. KEY POINTS: • Less and more efficient glucose utilization for suspension cell growth • Concerted alteration of metabolic enzyme activity and protein expression • Protein candidates to interfere glycolytic activity in MDCK cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00253-021-11150-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7907048PMC
March 2021

Single-Use Capture Purification of Adeno-Associated Viral Gene Transfer Vectors by Membrane-Based Steric Exclusion Chromatography.

Hum Gene Ther 2021 09 30;32(17-18):959-974. Epub 2021 Mar 30.

Center for Infectious Diseases, Virology, Heidelberg University Hospital, Heidelberg, Germany.

We present membrane-based steric exclusion chromatography (SXC) as a universal capture step for purification of adeno-associated virus (AAV) gene transfer vectors independent of their serotype and surface characteristics. SXC is performed by mixing an unpurified cell culture supernatant containing AAV particles with polyethylene glycol (PEG) and feeding the mixture onto a chromatography filter unit. The purified AAV particles are recovered by flushing the unit with a solution lacking PEG. SXC is an inexpensive single-use method that permits to concentrate, purify, and re-buffer AAV particles with yields >95% and >80% impurity clearance. SXC could theoretically be employed at industrial scales with units of nearly 20 m.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/hum.2019.284DOI Listing
September 2021

High cell density perfusion process for high yield of influenza A virus production using MDCK suspension cells.

Appl Microbiol Biotechnol 2021 Feb 30;105(4):1421-1434. Epub 2021 Jan 30.

State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.

Similar to the recent COVID-19 pandemic, influenza A virus poses a constant threat to the global community. For the treatment of flu disease, both antivirals and vaccines are available with vaccines the most effective and safest approach. In order to overcome limitations in egg-based vaccine manufacturing, cell culture-based processes have been established. While this production method avoids egg-associated risks in face of pandemics, process intensification using animal suspension cells in high cell density perfusion cultures should allow to further increase manufacturing capacities worldwide. In this work, we demonstrate the development of a perfusion process using Madin-Darby canine kidney (MDCK) suspension cells for influenza A (H1N1) virus production from scale-down shake flask cultivations to laboratory scale stirred tank bioreactors. Shake flask cultivations using semi-perfusion mode enabled high-yield virus harvests (4.25 log(HAU/100 μL)) from MDCK cells grown up to 41 × 10 cells/mL. Scale-up to bioreactors with an alternating tangential flow (ATF) perfusion system required optimization of pH control and implementation of a temperature shift during the infection phase. Use of a capacitance probe for on-line perfusion control allowed to minimize medium consumption. This contributed to a better process control and a more economical performance while maintaining a maximum virus titer of 4.37 log(HAU/100 μL) and an infectious virus titer of 1.83 × 10 virions/mL. Overall, this study clearly demonstrates recent advances in cell culture-based perfusion processes for next-generation high-yield influenza vaccine manufacturing for pandemic preparedness. KEY POINTS: • First MDCK suspension cell-based perfusion process for IAV produciton was established. • "Cell density effect" was overcome and process was intensified by reduction of medium use and automated process control. • The process achieved cell density over 40 × 10 cells/mL and virus yield over 4.37 log(HAU/100 μL).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00253-020-11050-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7847233PMC
February 2021

OP7, a novel influenza A virus defective interfering particle: production, purification, and animal experiments demonstrating antiviral potential.

Appl Microbiol Biotechnol 2021 Jan 4;105(1):129-146. Epub 2020 Dec 4.

Bioprocess Engineering, Otto von Guericke University Magdeburg, Magdeburg, Germany.

The novel influenza A virus (IAV) defective interfering particle "OP7" inhibits IAV replication in a co-infection and was previously suggested as a promising antiviral agent. Here, we report a batch-mode cell culture-based production process for OP7. In the present study, a seed virus containing standard virus (STV) and OP7 was used. The yield of OP7 strongly depended on the production multiplicity of infection. To inactivate infectious STV in the OP7 material, which may cause harm in a potential application, UV irradiation was used. The efficacy of OP7 in this material was preserved, as shown by an in vitro interference assay. Next, steric exclusion chromatography was used to purify and to concentrate (~ 13-fold) the UV-treated material. Finally, administration of produced OP7 material in mice did not show any toxic effects. Furthermore, all mice infected with a lethal dose of IAV survived the infection upon OP7 co-treatment. Thus, the feasibility of a production workflow for OP7 and its potential for antiviral treatment was demonstrated. KEY POINTS: • OP7 efficacy strongly depended on the multiplicity of infection used for production • Purification by steric exclusion chromatography increased OP7 efficacy • OP7-treated mice were protected against a lethal infection with IAV.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00253-020-11029-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7778630PMC
January 2021

Production of Modified Vaccinia Ankara Virus by Intensified Cell Cultures: A Comparison of Platform Technologies for Viral Vector Production.

Biotechnol J 2021 Jan 8;16(1):e2000024. Epub 2020 Sep 8.

Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, Sandtorstr. 1, Magdeburg, 39106, Germany.

Modified Vaccinia Ankara (MVA) virus is a promising vector for vaccination against various challenging pathogens or the treatment of some types of cancers, requiring a high amount of virions per dose for vaccination and gene therapy. Upstream process intensification combining perfusion technologies, the avian suspension cell line AGE1.CR.pIX and the virus strain MVA-CR19 is an option to obtain very high MVA yields. Here the authors compare different options for cell retention in perfusion mode using conventional stirred-tank bioreactors. Furthermore, the authors study hollow-fiber bioreactors and an orbital-shaken bioreactor in perfusion mode, both available for single-use. Productivity for the virus strain MVA-CR19 is compared to results from batch and continuous production reported in literature. The results demonstrate that cell retention devices are only required to maximize cell concentration but not for continuous harvesting. Using a stirred-tank bioreactor, a perfusion strategy with working volume expansion after virus infection results in the highest yields. Overall, infectious MVA virus titers of 2.1-16.5 × 10  virions/mL are achieved in these intensified processes. Taken together, the study shows a novel perspective on high-yield MVA virus production in conventional bioreactor systems linked to various cell retention devices and addresses options for process intensification including fully single-use perfusion platforms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/biot.202000024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7435511PMC
January 2021

Application of an Inclined Settler for Cell Culture-Based Influenza A Virus Production in Perfusion Mode.

Front Bioeng Biotechnol 2020 2;8:672. Epub 2020 Jul 2.

Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany.

Influenza viruses have been successfully propagated using a variety of animal cell lines in batch, fed-batch, and perfusion culture. For suspension cells, most studies reported on membrane-based cell retention devices typically leading to an accumulation of viruses in the bioreactor in perfusion mode. Aiming at continuous virus harvesting for improved productivities, an inclined settler was evaluated for influenza A virus (IAV) production using the avian suspension cell line AGE1.CR.pIX. Inclined settlers present many advantages as they are scalable, robust, and comply with cGMP regulations, e.g., for recombinant protein manufacturing. Perfusion rates up to 3000 L/day have been reported. In our study, successful growth of AGE1.CR.pIX cells up to 50 × 10 cells/mL and a cell retention efficiency exceeding 96% were obtained with the settler cooled to room temperature. No virus retention was observed. A total of 5.4-6.5 × 10 virions were produced while a control experiment with an ATF system equaled to 1.9 × 10 virions. For infection at 25 × 10 cells/mL, cell-specific virus yields up to 3474 virions/cell were obtained, about 5-fold higher than for an ATF based cultivation performed as a control (723 virions/cell). Trypsin activity was shown to have a large impact on cell growth dynamics after infection following the cell retention device, especially at a cell concentration of 50 × 10 cells/mL. Further control experiments performed with an acoustic settler showed that virus production was improved with a heat exchanger of the inclined settler operated at 27°C. In summary, cell culture-based production of viruses in perfusion mode with an inclined settler and continuous harvesting can drastically increase IAV yields and possibly the yield of other viruses. To our knowledge, this is the first report to show the potential of this device for viral vaccine production.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fbioe.2020.00672DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7343718PMC
July 2020

Synthesis of lipid-linked oligosaccharides by a compartmentalized multi-enzyme cascade for the in vitro N-glycosylation of peptides.

J Biotechnol 2020 Oct 9;322:54-65. Epub 2020 Jul 9.

Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, Sandtorstr. 1, 39106 Magdeburg, Germany; Otto-von-Guericke University Magdeburg, Chair of Bioprocess Engineering, Postfach 4120, 39106 Magdeburg, Germany. Electronic address:

A wide range of glycoproteins can be recombinantly expressed in aglycosylated forms in bacterial and cell-free production systems. To investigate the effect of glycosylation of these proteins on receptor binding, stability, efficacy as drugs, pharmacodynamics and pharmacokinetics, an efficient glycosylation platform is required. Here, we present a cell-free synthetic platform for the in vitro N-glycosylation of peptides mimicking the endoplasmic reticulum (ER) glycosylation machinery of eukaryotes. The one-pot, two compartment multi-enzyme cascade consisting of eight recombinant enzymes including the three Leloir glycosyltransferases, Alg1, Alg2 and Alg11, expressed in E. coli and S. cerevisiae, respectively, has been engineered to produce the core lipid-linked (LL) oligosaccharide mannopentaose-di-(N-acetylglucosamine) (LL-Man5). Pythanol (CHO), a readily available alcohol consisting of regular isoprenoid units, was utilized as the lipid anchor. As part of the cascade, GDP-mannose was de novo produced from the inexpensive substrates ADP, polyphosphate and mannose. To prevent enzyme inhibition, the nucleotide sugar cascade and the glycosyltransferase were segregated into two compartments by a cellulose ester membrane with 3.5 kDa cut-off allowing for the effective diffusion of GDP-mannose across compartments. Finally, as a proof-of-principle, pythanyl-linked Man5 and the single-subunit oligosaccharyltransferase Trypanosoma brucei STT3A expressed in Sf9 insect cells were used to in vitro N-glycosylate a synthetic peptide of ten amino acids bearing the eukaryotic consensus motif N-X-S/T.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jbiotec.2020.07.003DOI Listing
October 2020

Impact of feeding and stirring regimes on the internal stratification of microbial communities in the fermenter of anaerobic digestion plants.

Bioresour Technol 2020 Oct 13;314:123679. Epub 2020 Jun 13.

Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, Sandtorstraße 1, 39106 Magdeburg, Germany. Electronic address:

In anaerobic digestion plants (ADs), homogenization of the feed, fermenter content and microbial communities is crucial for efficient and robust biogas production. However, mixing also requires a significant amount of energy. For an 850 m agricultural AD equipped with eight sampling ports, we investigated whether different feeding and stirring regimes enable a sufficient homogenization of the microbial community using metaproteomics and terminal restriction fragment length polymorphism (TRFLP) analysis. Systematic comparison of samples taken at the top and the bottom as well as at the rim and the center of the AD using scatter plots and students t-test revealed only a small number of differences in metaproteins, taxonomies and biological processes. Obviously, the applied stirring and feeding conditions were sufficient to largely homogenize the content of the AD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biortech.2020.123679DOI Listing
October 2020
-->